The goal of the proposed research is the development of novel approaches to ameliorate erythrocyte sickling in individuals with sickle cell disease (SCD). Although this disease was discovered more than a century ago, we still lack effective mechanism-based therapies. This disease is the most prevalent inherited genetic disorder and affects millions worldwide. As an important mission of NIH is to develop highly innovative treatments for this disease, the goal of the proposed work should be of high priority. Research proposed here is based on unexpected discoveries resulting from an unbiased, high-throughput metabolomic screen that identified several metabolites including adenosine (Ado), sphingosine-1-phosphate (S1P) and 2,3- diphosphoglycerate (2,3-DPG) that are highly elevated in the blood of mice and humans with SCD. Follow-up studies showed that these three elevated metabolites collaboratively work together to promote sickling, the central pathogenic process of the disease. The project has interrelated goals to translate our findings into innovative therapeutics for SCD by providing new insight into disease pathogenesis. In AIM I, we propose to use both pharmacological and genetic approaches to determine whether excess Ado signaling via A2BR induces 2,3-DPG production by protein kinase A (PKA)-dependent phosphorylation of key enzymes involved in 2,3-DPG generation. These studies will provide novel mechanism underlying Ado signaling-mediated 2,3-DPG induction and add a new chapter to erythrocyte physiology and pathology. In AIM II, we will explore different approaches including genetic, pharmacological, cellular, biochemical, and high throughput robotic co- crystallography coupled with spectral functional analysis to determine how S1P: 1) functions as an intracellular allosteric modulator to regulate Hb-O2 affinity and 2) functions via extracellular S1P receptors to induce inflammation. Additional experiments encompass a set of preclinical studies to test the efficacy and safety of lowering S1P by specific antagonists in SCD mice. These studies are expected to provide the foundation for appropriate clinical trials in humans with SCD. Third, the molecular basis underlying elevated S1P inside RBCs and in plasma will be investigated. Briefly, we propose to test a novel hypothesis that A2BR functions upstream to induce S1P production within RBCs and that activated platelets and complement activation-induced intravascular hemolysis underlie the further elevation of S1P in plasma. In AIM IV, we propose translational studies to conduct an analysis of approximately 235 adult blood samples obtained from a large cohort of SCD patients by collaborating with the NHLBI to determine whether these newly identified elevated metabolites are pathogenic biomarkers correlating to disease severity and phenotypic variation. Overall, the proposed preclinical animal studies and translational human studies are expected to reveal important therapeutic targets in the medical management of SCD patients.